Bacteroides fragilis is the most frequent opportunistic pathogen isolated from anaerobic infections. The success of B. fragilis pathogenicity is still not completely understood but virulence factors such as capsular polysaccharides, adherence, and neuraminidase play an important role. Potential virulence factor that has yet to be characterized is the ability of B. fragilis to produce hemolysins/cvtolysins. To date, no hemolysin/cytolysin has been characterized in this anaerobic pathogen despite the fact that some clinical isolates show a hemolytic phenotype on blood agar plates. Analysis of the complete B. fragilis genome sequence revealed the presence of [ten] genes homologous to bacterial hemolysins supporting our idea that hemolysins might be involved in the pathogenicity of B. fragilis. Hemolysins are cytotoxic proteins produced by many organisms ranging from prokaryotes to eukaryotes. Though they are named for their capacity to lyse erythrocytes, many are toxic to other cell types as well. Microbial hemolysins are considered to be virulence factors that provide an advantage to invading microorganisms by their ability to kill incoming leukocytes. This may enhance survival by weakening the host immune response and gaining access to nutrients. Despite this extended number of genes homologous to hemolysins, no study has characterized the role and function of hemoiysins/cytolysins in B. fragilis pathogenicity. Thus, the long-term goal of this research project is to test the hypothesis that hemolysins/cytolysins are virulence factors in B. fragilis pathogenicity. In the present study we propose to initiate studies to identify and characterize the genes conferring hemolytic activity in B. fragilis and delineate the role of hemolysins in experimental infection and neutrophil toxicity. To accomplish these objectives, the following aims will be pursued. [Aim 1. Delineate the role of B. fragilis hemolysins in experimental infections.] A) We will assess the expression of hemolysin genes in vivo by Real-Time RT-PCR. B) Delineate the immune response to hemolysins as a mean to evaluate their expression in vivo. C) Elucidate the role of hemolysins in an intra-abdominal abscess model. D) Determine the toxicity of hemolysins to PMNs. [Aim 2. Complete the analysis of B. fragilis hemolysins.] A) We will complete the initial analysis of the hemolytic/cytolytic activity of B. fragilis hemolysins. B) We will complete the construction of hemolysin mutants. [unreadable] [unreadable] [unreadable]